Diagnosis of meningitis in immunocompromised hosts, such as those with AIDS or cancer, is difficult because of the unavailability of sensitive methods for detecting the most likely pathogens. Organisms in these settings, such as cytomegalovirus (CMV), Herpes simplex virus (HSV) and Varicella-Zoster virus (VZV), are difficult to grow from cere-brospinal fluid (CSF), and no good antigen detection techniques are available. Over the past few years, it has become increasingly clear that detecting these viral agents by the polymerase chain reaction (PCR) method may provide the most sensitive and specific diagnostic tests. Therefore, we have undertaken development of a battery of in-house PCR assays for CMV, HSV, and VZV so they will be available as routine service tests for CSF from NIH patients. In 1997 development and clinical validation of the PCR assay for CMV in spinal fluid was completed and the test began to be offered as a routine molecular diagnostic assay. During 1998 final development and validation of the PCR assay for HSV in spinal fluid was completed and the test is now also offered as a routine molecular diagnostic assay. The HSV assay on spinal fluid will specifically determine the presence of either HSV1, HSV2, or both viral DNAs. Development of a VZV assay with the same performance characteristics as the existing CMV and HSV assays will continue. All three assays are designed to utilize internal mimics to evaluate the efficiency of amplification within individual PCR tubes and to detect PCR inhibition, in order to eliminate this source of false negative results. Endpoint detection by europium-labeled fluorescence hybridization probes allows a lower limit of detection of three to five viral genome equivalents. Evaluations of the CMV and HSV assays show excel-lent specificity. The sensitivity and reliability of molecular diagnostic techniques depends not only on the efficiencies of the amplification and detection methods utilized, but also on the performance of the sample preparation methods used. In the past year a large-scale project was undertaken to investigate the relative efficiencies and reliabilities of six different methods for preparing nucleic acids from human spinal fluid, respiratory samples or blood. We have identified a sample preparation method that appears to be highly reliable and offers improved efficiency of DNA recovery. This method has now been incorporated into the clinical molecular diagnostic assays offered.